Structure and stability of the N-terminal domain of the ribosomal protein L9: evidence for rapid two-state folding.
The N-terminal domain, residues 1-56, of the ribosomal protein L9 has been chemically synthesized. The isolated domain is monomeric as judged by analytical ultracentrifugation and concentration-dependent CD. Complete 1H chemical shift assignments were obtained using standard methods. 2D-NMR experiments show that the isolated domain adopts the same structure as seen in the full-length protein. It consists of a three-stranded antiparallel beta-sheet sandwiched between two helixes. Thermal and urea unfolding transitions are cooperative, and the unfolding curves generated from different experimental techniques, 1D-NMR, far-UV CD, near-UV CD, and fluorescence, are superimposable. These results suggest that the protein folds by a two-state mechanism. The thermal midpoint of folding is 77 +/- 2 degrees C at pD 8.0, and the domain has a delta G degree folding = 2.8 +/- 0.8 kcal/mol at 40 degrees C, pH 7.0. Near the thermal midpoint of the unfolding transition, the 1D-NMR peaks are significantly broadened, indicating that folding is occurring on the intermediate exchange time scale. The rate of folding was determined by fitting the NMR spectra to a two-state chemical exchange model. Similar folding rates were measured for Phe 5, located in the first beta-strand, and for Tyr 25, located in the short helix between strands two and three. The domain folds extremely rapidly with a folding rate constant of 2000 s-1 near the midpoint of the equilibrium thermal unfolding transition.
Kuhlman
B
B
Department of Chemistry, State University of New York, Stony Brook 11794-3400, USA.
Boice
J A
JA
Fairman
R
R
Raleigh
D P
DP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects; D002500:Centrifugation, Isopycnic; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus; D006358:Hot Temperature; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C049650:ribosomal protein L9; D014508:Urea